Bifunctionalized polyester material for surface treatment and biomodification

ABSTRACT

The invention features a method of treating polyester material to generate functional carboxylic acid and amine groups. These functional groups can be used as sites for covalent bond formation to attach chemical or biological moieties. This bifunctionalized polyester polymer can be used in any medical application in which biocompatible polymers are used.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/352,286, filedJan. 27, 2003 (currently pending), which claims benefit of 60/351,760,filed Jan. 25, 2002; each application is hereby incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported by the Government under Grant No. R01 HL21796from the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The invention relates to modification of polyester materials.

BACKGROUND OF THE INVENTION

Polyester (DACRON® or polyethylene terephthalate) fibers were firstcharacterized in 1941 and have become the most widely produced syntheticfiber in the world. They are most familiarly known by the DuPontcommercial name DACRON®. The polymer is synthesized by a condensationreaction of derivatives of ethylene glycol and terephthalic acid,resulting in molecules that contain 80 to 100 repeat units. Thesemolecules are then extruded through a plurality of holes (a spinneret)to produce multi-filament fibrous filaments. Such DACRON® fibers arefurther processed into various structures such as warp-knit, weft-knit,and woven fabrics that have excellent resiliency as well as resistanceto a wide range of chemical and biological challenges.

DACRON® is utilized in items ranging from clothing to medical implants.DACRON® yarn was first sewn into a tubular form and utilized as alarge-diameter vascular graft in the mid-1950s. Since that point,DACRON® has been incorporated into both large and medium bore vasculargrafts in knitted and woven form. These grafts have shown excellentlong-term biodurability, handling characteristics and capsular tissueincorporation.

Polyester is known as a relatively inert fiber. It is hydrophobic, bothin bulk and in its surface properties. At normal temperatures it has lowuptake of moisture, dyes and other chemicals. In normal textile use, ittends to suffer associated disadvantages: it generates staticelectricity, it does not readily shed oily soils, and it does not wetenough to encourage the wicking of water. For applications whererepellency is required, however, it is insufficiently hydrophobic, andrepellent finishes are applied. Like any fiber, softness isadvantageous, and chemical softeners are applied.

Modifications to overcome these deficiencies typically rely on thesurface deposition of polymeric textile finishes. These includesilicones, vinyl and acrylic polymers, and fluorochemicals. Otherfinishes are based on an ester interchange reaction that fixes ahydrophilic moiety (typically a short chain polyethylene oxide). Many ofthese finishes suffer a lack of durability to laundering and drycleaning, since (other than those bonded via ester interchange) they arenot covalently bonded to the polyester surface.

Polyester is employed for various medical devices such as prostheticvascular grafts, prosthetic heart valve sewing cuffs, left ventricularassist devices, artificial organs, wound patches and wound dressings.Polyester is a biodurable material due to the relatively inertproperties of the polymer and can persist for greater than 10 years whenimplanted without significant deleterious effects to the specificdevice. However, this material, similar to other biomaterials, is proneto 3 major complications when implanted: 1) thrombosis (clot formation),2) infection and 3) lack of cell appropriate healing. These adverseproperties occur as a result of the bulk properties of the polymer.Additionally, the rigidity of the polymer limits surface modification inorder to incorporate various moieties such as anti-thrombolytic agents(e.g., anti-thrombin), thrombolytic agents, growth-promoting factors,growth-inhibiting factors, and antimicrobial/antifungal agents.

A complication of all implantable biomaterials is incompatibilitybetween blood and the biomaterial surface. The initial interaction ofblood and the foreign surface results in an array of activation orbiologic responses: platelet activation and adhesion, activation of theintrinsic pathway of the coagulation cascade resulting in formation ofactive thrombin, leukocyte activation and the release of complement andkallikrein. If unregulated, these responses lead to surface thrombusformation with subsequent failure of the implanted biomaterial.

Numerous attempts have been made to create a more biocompatible surfaceby establishing a new biologic lining on the luminal surface that would“passivate” this acute initial reaction. These efforts have ranged fromnon-specifically binding albumin to the surface followed by heatdenaturation to non-specifically crosslinking albumin, gelatin andcollagen. Covalent or ionic binding of the anticoagulant heparin alone,in conjunction with other biologic compounds, or with spacer moieties aswell as covalent linkage of thrombomodulin have also been performed.Other studies have focused on modifying the composition of thebiomaterial by either increasing hydrophilicity via incorporation ofpolyethylene oxide groups or by creating an ionically charged surface.

Each of these methodologies has had limited success in creating adurable, biologically-active surface. There are several limitationsassociated with these surface modifications: 1) thrombin is not directlyinhibited therefore fibrinogen amounts remain constant on the materialsurface permitting platelet adhesion, 2) heparin-coated biomaterials maybe subject to heparitinases limiting long-term use of these materials,3) non-specifically bound compounds are desorbed from the surface whichis under shear stresses thereby re-exposing the thrombogenic biomaterialsurface, 4) rapid release of non-specifically bound compounds may createan undesired systemic effect and 5) charge-based polymers may be coveredby other blood proteins such that anticoagulant effects are masked.

Endothelial cells play a pivotal role in mediating blood interactionwith the arterial wall. These cells maintain hemostasis and alsosynthesize growth mediators that block abnormal smooth muscle cellproliferation. Ideally, prosthetic grafts should promote endothelialcell adherence and growth on the luminal surface while permitting directhost tissue incorporation at the capsular surface. This type of cellularincorporation does not occur in actuality, thereby predisposing thesegrafts to infection, thrombosis, perigraft seromas and delayed graftfailure. Thus, failure of appropriate cell type growth and developmentto these biomaterials significantly limits their expanded use.

To avert these complications and mimic the non-thrombogenic in vivoendothelial cell blood vessel lining, cell adhesion to prosthetic graftsusing endothelial cell seeding techniques have been extensivelyemployed. Adhesive proteins such as fibronectin, fibrinogen, vitronectinand collagen have served well in graft seeding protocols. The cellattachment properties of these matrices can also be duplicated by shortpeptide sequences such as RGD (Arg-Gly-Asp). These adhesive proteins,however, have several drawbacks: 1) bacterial pathogens recognize andbind to these sequences, 2) non-endothelial cell lines also bind tothese sequences, 3) patients requiring a seeded vascular graft have fewdonor endothelial cells, therefore cells must be grown in culture and 4)endothelial cell loss to shear forces remains a significant obstacle.

Modification of the surface has also been employed to modify hostresponse to the foreign body, serving as an approach for improvingendothelial cell adherence to DACRON®. Endothelial cells after seedinghave been shown to attach and grow on a variety of protein substratescoated onto vascular graft materials. Bioactive oligopeptides and cellgrowth factors have been immobilized onto various polymers anddemonstrated to affect cell adherence and growth. Additional studieshave described the incorporation of growth factors into a degradableprotein mesh, resulting in the formation of capillaries into the graftwall. Utilizing these techniques to incorporate growth factors, however,does have major limitations: 1) growth factor is rapidly released fromthe matrix, 2) matrix degradation re-exposes the thrombogenic surface,thus endothelialization is not uniform and 3) release of non-endothelialspecific growth factor is not confined to the biomaterial matrix,thereby exposing the “normal” distal artery to the growth factor.

There have been several studies assessing the effects of amineinteraction with polyester. Zahn et al. (Polymer 3:429, 1962), as wellas Farrow et al. (Polymer 3:17, 1962) assessed the lysis of polyester inan attempt to breakdown excess material in the textile industry intosmaller components, without regard to maintaining the integrity of thepolymer structure. In 1982, Ellison et. al. examined the effects of amonofunctional amine versus alkali hydrolysis on polyester. Thesestudies, which again were performed under harsh conditions, demonstratedthat alkaline hydrolysis has a more substantial effect on fiber weightwithout extensive strength loss. In contrast, aminolysis had less effecton fiber weight but a greater effect on fiber strength, indicative of apermanent reaction within the polymer structure. In 1968, Avny andRebenfeld demonstrated that multi-functional amine compounds could bereacted within the polymer structure (three or more amine groups) whilepresenting minimal loss in strength (Applied Polymer Science 32:4009,1986). There remains a need, however, for a polyester material thatprovides functional moieties for attachment of commercial finishes orbiologically-active agents, while retaining material strength.

SUMMARY OF THE INVENTION

This invention features a method of generating a functionalizedpolyester material. This method includes incubating the polyestermaterial with ethylene diamine in solution (aqueous or organic) underconditions that result in functionalization of the polyester material.The functionalization of the material consists of creation of carboxylicacid and amine groups within the polyester backbone. The carboxylic acidand amine groups can then be used as sites to attach other chemicalcompounds and biologically-active agents to the polyester backbone viaan ionic or covalent bond.

In a desired embodiment, the chemical compound consists of a commercialfinish selected from the group consisting of flame retardants,repellents, antistatic agents, and dyes.

In several desired embodiments, the biologically-active agent applied tothe bifunctionalized polyester polymer is desirably a small molecule(e.g., an organic compound with a mw<1000), but can also include, forexample, a peptide, a polypeptide, a protein, a nucleic acid molecule,or an antibody. The biologically-active agent can act as anantimicrobial agent, an antifungal agent, an anti-thrombolytic agent(e.g., anti-thrombin), a thrombolytic agent, an antiviral agent, anantiseptic agent, an antibiotic, a growth-inhibiting agent, agrowth-promoting agent, or a combination thereof. The antibiotic used inthe method can include quinolone. Inorganic therapeutically-activecompounds such as silver, silver salts, gold, or gold salts may also bebonded to the polymers of the present invention. This bonding mayinvolve a covalent or an ionic interaction between the compound and thecarboxylic acid group or amine group of the bifunctionalized polyesterpolymer.

The bifunctionalized polyester polymer which has bound an effectiveamount of the therapeutic compound or biologic agent can be used in anymedical application in which biocompatible polymers are used (e.g., abiocompatible device), and in which infection or other complications areto be avoided. Examples include, but are not limited to, use as a wounddressing or implantable device. Desired devices are catheters, vasculargrafts, artificial hearts, other artificial organs and tissues, bloodfilters, pacemaker leads, heart valves, and prosthetic grafts. Thebifunctional polyester material, when used in vascular grafts, shouldnot activate coagulation or-inhibit cellular healing, is desirablybiodurable, non-thrombogenic, chemically durable, resistant to infectionor formation of microbial plaques, easy to implant, and possessesappropriate elastic properties. The bifunctional polyester materialshould also be sufficiently malleable so that it can form theappropriate geometry, but also have sufficient tensile strength toendure rigorous circulation throughout the vascular tree. The surfaceproperties of the graft can be modified with biologically-activeproteins in order to emulate certain natural properties of nativevessels, thereby improving graft patency and healing. For instance,anti-thrombin (recombinant hirudin) or other anti-clotting agents,thrombolytic agents (e.g. streptokinase, urokinase, tissue plasminogenactivator (tPA), pro-urokinase, etc.), and mitogenic agents (e.g.vascular endothelial growth factor) or other growth promotingsubstances, or inhibitors (e.g. γ-interferon) can be linked to thesurface of the graft.

The biocompatible material should be able to be sterilized, for example,by gamma radiation.

In various embodiments, the bifunctionalized polyester polymer isnon-toxic, does not contain an exogenous binder agent, and/or does notinduce clot formation. The bifunctionalized polyester polymer can alsobe used in commercial products that are desirably antibacterial,antiviral, or antifungal (e.g., shower curtains, clothing, and foamcushions).

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a schematic showing the hydrolysis and aminolysis ofpolyester.

FIGS. 2A-2G are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of methylene blueindicates the presence of carboxylic acid groups. Polyester segments aretreated with 100% EDA for 80 minutes, rinsed in water for 5 min. (FIG.2A), 10 min. (FIG. 2B), 20 min. (FIG. 2C), 40 min. (FIG. 2D), 120 min.(FIG. 2E), 240 min. (FIG. 2F), or overnight (16.25 hours; FIG. 2G), andexposed to methylene blue at pH 8.

FIGS. 3A-3G are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of acid redindicates the presence of amine groups. Polyester segments are treatedwith 100% EDA for 80 minutes, rinsed in water for 5 min. (FIG. 3A), 10min. (FIG. 3B), 20 min. (FIG. 3C), 40 min. (FIG. 3D), 120 min. (FIG.3E), 240 min. (FIG. 3F), or overnight (16.25 hours; FIG. 3G), andexposed to acid red at pH 4.

FIGS. 4A-4E are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of methylene blueindicates the presence of carboxylic acid groups. Polyester segments aretreated with 100% EDA (FIG. 4A), 90% EDA in water (FIG. 4B), 80% EDA inwater (FIG. 4C), 70% EDA in water (FIG. 4D), or 50% EDA in water (FIG.4E) for 80 minutes, rinsed in water for 10 min., and exposed tomethylene blue at pH 8.

FIGS. 5A-5E are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of acid redindicates the presence of amine groups. Polyester segments are treatedwith 100% EDA (FIG. 5A), 90% EDA in water (FIG. 5B), 80% EDA in water(FIG. 5C), 70% EDA in water (FIG. 5D), or 50% EDA in water (FIG. 5E) for80 minutes, rinsed in water for 10 min., and exposed to acid red at pH4.

FIGS. 6A-6F are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of methylene blueindicates the presence of carboxylic acid groups. Polyester segments aretreated with 100% EDA (FIG. 6A), 90% EDA in toluene (FIG. 6B), 80% EDAin toluene (FIG. 6C), 70% EDA in toluene (FIG. 6D), 50% EDA in toluene(FIG. 6E), or 25% EDA in toluene (FIG. 6F), for 80 minutes, rinsed inwater for 10 min., and exposed to methylene blue at pH 8.

FIGS. 7A-7F are a series of photographs of scoured ethylenediamine-treated polyester segments in which the uptake of acid redindicates the presence of amine groups. Polyester segments are treatedwith 100% EDA (FIG. 7A), 90% EDA in toluene (FIG. 7B), 80% EDA intoluene (FIG. 7C), 70% EDA in toluene (FIG. 7D), 50% EDA in toluene(FIG. 7E), or 25% EDA in toluene (FIG. 7F), for 80 minutes, rinsed inwater for 10 min., and exposed to acid red at pH 4.

FIG. 8A-8F are a series of photographs of hydrolysed ethylenediamine-treated polyester segments in which the uptake of methylene blueindicates the presence of carboxylic acid groups. Polyester segments aretreated with 100% EDA (FIG. 8A), 90% EDA in toluene (FIG. 8B), 80% EDAin toluene (FIG. 8C), 70% EDA in toluene (FIG. 8D), 50% EDA in toluene(FIG. 8E), or 25% EDA in toluene (FIG. 8F), for 80 minutes, rinsed inwater for 10 min., and exposed to methylene blue at pH 8.

FIGS. 9A-9F are a series of photographs of hydrolysed ethylenediamine-treated polyester segments in which the uptake of acid redindicates the presence of amine groups. Polyester segments are treatedwith 100% EDA (FIG. 9A), 90% EDA in toluene (FIG. 9B), 80% EDA intoluene (FIG. 9C), 70% EDA in toluene (FIG. 9D), 50% EDA in toluene(FIG. 9E), or 25% EDA in toluene (FIG. 9F), for 80 minutes, rinsed inwater for 10 min., and exposed to acid red at pH 4.

FIGS. 10A-10G are a series of photographs of ethylene diamine-treatedpolyester segments in which the uptake of methylene blue indicates thepresence of carboxylic acid groups. Polyester segments are treated with100% EDA for 80 minutes, rinsed in toluene for 5 min. (FIG. 10A), 10min. (FIG. 10B), 20 min. (FIG. 10C), 40 min. (FIG. 10D), 120 min. (FIG.10E), 240 min. (FIG. 10F), or overnight (16.25 hours; FIG. 10G), andexposed to methylene blue at pH 8.

FIGS. 11A-11G are a series of photographs of ethylene diamine-treatedpolyester segments in which the uptake of acid red indicates thepresence of amine groups. Polyester segments are treated with 100% EDAfor 80 minutes, rinsed in toluene for 5 min. (FIG. 11A), 10 min. (FIG.11B), 20 min. (FIG. 11C), 40 min. (FIG. 11D), 120 min. (FIG. 11E), 240min. (FIG. 11F), or overnight (16.25 hours; FIG. 110G), and exposed toacid red at pH 4.

FIG. 12 is a photograph showing the uptake of CI Acid Red 1 or MethyleneBlue dye after treatment of polyester fabric (hydrolyzed orunhydrolyzed) at 85° C. with 2-methylpentamethylene diamine (2MPD) for10 minutes, tetraethylenepentamine (TEP) for 20 minutes,1,2-diaminocyclohexane (12DC) for 2 hours, and 1,6-hexanediamine (16HD)for 24 hours.

FIG. 13 is a photograph showing the uptake of CI Acid Red 1 dye aftertreatment of polyester fabric with varying concentrations of EDA intoluene for 20 hours at 50° C.

FIG. 14 is a photograph showing the uptake of Acid Red 1 or MethyleneBlue dye into hydrolyzed or unhydrolyzed polyester fabric as aconsequence of immersion time in EDA.

FIGS. 15A and 15B are graphs showing the concentration of amino (FIG.15A) and carboxylic acid (FIG. 15B) groups after EDA treatment.

FIGS. 16A-16D are photographs at either 500×, 850×, 1500× or 2000×magnification, respectively, showing the cracking of polyester fibers.

FIG. 17 is a graph showing the loss of tensile strength of EDA-treatedpolyester.

FIGS. 18A and 18B are graphs showing the wicking performance ofEDA-treated polyester by weight (FIG. 18A) and by height (FIG. 18B).

FIG. 19 is a drawing of C.I. Acid Yellow 4 dye.

FIG. 20 is a graph showing the binding of ¹²⁵I-BSA to EDA-modifiedDACRON® using amine groups.

FIG. 21 is a graph showing the binding of ¹²⁵I-BSA to EDA-modifiedDACRON® using carboxylic acid groups.

FIG. 22 is a graph showing the binding of ¹²⁵I-BSA and of ¹³¹I-BSA toEDA-modified DACRON® using both amine and carboxylic acid groups.

DETAILED DESCRIPTION

The present invention features the use of a bifunctional amine compound,ethylene diamine (EDA), under select conditions (i.e., solution type,concentration, surface treatment) to establish both amine functionalgroups and carboxylic acid groups within the polyester backbone. Thegroups can then be reacted with various crosslinking agents, binders,catalysts or via direct ionic interaction with other moieties such ascommercial finishes (i.e., flame retardants, repellents, anti-staticagents, dyes) or biologically-active agents (i.e. anti-thrombolyticagents (e.g., anti-thrombin), thrombolytic agents,growth-promoting/inhibiting agents, antimicrobial or antifungal agents).The bifunctionalized polyester fiber can be provided for use in medicaland textile applications.

Binding to Functional Groups

The treatment of polyester using this method establishes a bifunctionalsurface onto which various agents, such as growth factors,anti-thrombolytic agents (e.g., anti-thrombin), thrombolytic agents, orantibiotics, can be bound by ionic or covalent interactions, eitheralone or in combination. Several methods can be employed for applicationof various finishes and biologic moieties to the surface. For finishes,functional groups capable of reacting with amine moieties include epoxy,isocyanate, methylol, fluoro- and chlorotriazine, vinyl sulphone and thelike. These reactive groups can be attached to substances capable ofincreasing or decreasing the hydrophobic nature of the polyestersurface, and thus modifying the static, wicking, softness and repellencyproperties of the fiber. Because these substances are thereby covalentlybonded to the polyester, their durability is substantially increased.For biologic moieties such as anti-thrombolytic agents (e.g.,anti-thrombin), thrombolytic agents, and growth-promoting/inhibitingagents, various crosslinking techniques employing homo- andheterobifunctional crosslinkers can be utilized. Additionally, variousinorganic and organic catalysts, reactive agents (i.e. glutaraldehyde)or binder agents can be used. Lastly, the ionic or hydrophilicproperties of the material could be exploited to incorporate variousantimicrobial/antifungal agents or biologic agents to the surface.

The presence of reactable amine groups on the polyester surface permitsattachment thereto of a range of materials. Lewin et. al. outlined someof the possible reactions and functionalities: functional groups capableof reacting with amine moieties include epoxy, isocyanate, methylol,fluoro- and chlorotriazine, vinyl sulphone and the like (Lewin et al.,Handbook of fiber science and technology, Vol. 2 Part B, New York,Marcel Dekker Inc. 1984). These reactive groups can be attached tosubstances capable of increasing or decreasing the hydrophobic nature ofthe polyester surface, and thus modifying the static, wicking, softnessand repellency properties of the fiber. Because these substances arethereby covalently bonded to the polyester, their durability issubstantially increased.

Finishes & Lubricants

Ranges of chemical finishes are available for spinning, weaving,knitting, and braiding productivity, as well as functional properties.They combine low fiber to metal frictional properties, good inter-fibercohesion, and excellent anti-static properties to maximize fiber,filament or yarn performance. For example 16-20% Lurol NF-782 aqueousemulsion spin finish is recommended for fine denier filament yarns suchas polyester with 0.8-1.2% take up on the weight of the yarn. Theemulsion is prepared by adding the finish slowly into rapidly agitating45-50° C. water. The emulsion should be translucent; opalescent inconcentrations up to 20%. Typical properties include a clear yellowappearance of the liquid at 25° C., gardner color <1, Viscosity cSt 56and pH of 8.2 in 5% aqueous solution. It begins to freeze if storedbelow 10° C. If frozen, the product should be warmed above 25° C. andstirred before use to insure homogeneity. Some bactericide should beadded to the emulsion to assure adequate storage life.

Finish level can be measured by conventional solvent extractiontechniques, e.g., using a blend of isopropanol and n-hexane as solventsfor polyester.

The spinning finishes according to the invention may containemulsifiers, wetting agents and/or antistatic agents and, optionally,standard auxiliaries, such as pH regulators, filament compacting agents,bactericides, and conductive polymers. Suitable emulsifiers, wettingagents and/or antistatic agents are anionic, cationic and/or nonionicsurfactants, such as mono- and/or diglycerides, for example glycerol,mono- and/or dioleate, alkoxylated, preferably ethoxylated and/orpropoxylated, fats, oils, fatty alcohols, castor oil containing 25 molethylene oxide (EO) and/or 16-18 fatty alcohol containing 8 molpropylene oxide and 6 mol EO, if desired alkoxylated 8-24 fatty acidmono- and/or diethanolamides, for example optionally ethoxylated oleicacid mono- and/or diethanolamide, tallow fatty acid mono- and/ordiethanolamide and/or coconut oil fatty mono- and/or diethanolamide,alkali metal and/or ammonium salts of alkoxylated, preferablyethoxylated and/or propoxylated, optionally end-capped 8-22 alkyl and/or8-22 alkylene alcohol sulfonates, reaction products of optionallyalkoxylated 8-22 alkyl alcohols with phosphorus pentoxide or phosphorusoxychloride in the form of their alkali metal, ammonium and/or aminesalts, for examples phosphoric acid esters of ethoxylated 12-14 fattyalcohols, neutralized with alkanolmine, alkali metal and/or ammoniumsalts of 8-22 alkyl sulfosuccinates, for example sodium dioctylsulfosuccinate and/or amine oxide, for example dimethyl dodecyl amineoxide. In considering this list of examples, it is important to bear inmind that many of the substances mentioned can perform not just onefunction, but several functions. Thus, an antistatic agent may also actas an emulsifier.

Suitable filament compacting agents are the polyacrylates, fatty acidsarcosides and/or copolymers with makeic anhydride known from the priorart (Melliand Textilberichte (1977), page 197), polyurethanes accordingto DE-A-38 30 468, pH regulators (e.g., C₁₋₄ carboxylic acids and/orC₁₋₄ hydroxycarboxylic acids (e.g., acetic acid and/or glycolic acid)),alkali metal hydroxides (e.g., potassium hydroxide), amines (e.g.,triethanolamine), and bactericides.

UV Inhibitors

Ranges of commercially available high performance fibers are susceptibleto ultra violet (UV) exposure. A list of typical stabilizers against UV,both radical formation & biodegradation, includes:2-hydroxybenzophenones, 2-hydroxypenyl-2-(2H)-benzotriazoles,cinnamates, and mixtures thereof. These chemicals are capable ofabsorbing and dissipating UV energy, thus inhibiting UV degradation.Free radicals are neutralized by hindered amine light stabilizers(HALS), butylated hydroxyanisole (BHA) and butylated hydroxytoluene(BHT).

Antimicrobials

Antimicrobials include silver nitrate, iodized radicals (e.g., TRIOSYN®;Hydro Biotech), benzylalkonium chloride and alkylpyridinium bromide(cetrimide) or alkyltrimethylammonium bromide. It is within the scope ofthis disclosure to coat or impregnate the bifunctionalized polyestermaterial disclosed herein as well as implants and prosthetic devicesmade therefrom with one or more materials which enhance itsfunctionality, e.g., surgically useful substances, such as those whichaccelerate or beneficially modify the healing process when the materialis implanted within a living organism. Thus, for example, antimicrobialagents such as broad spectrum antibiotics (gentamicin sulphate,erythromycin, or derivatized glycopeptides), which are slowly releasedinto the tissue, can be incorporated to aid in combating clinical andsubclinical infections in a surgical or trauma wound site. Otherantimicrobials that can be used in the compositions of the inventioninclude those described in U.S. Pat. Nos. 6,013,106; 6,464,971;6,465,429; 6,471,974; 6,472,384; 6,472,424; 6,475,771; 6,479,454;6,485,928; 6,492,328; 6,500,861; 6,506,737; 6,509,349; 6,436,445;6,426,369; 6,423,748; 6,420,116; 6,417,217; 6,407,288; 6,387,928; and6,376,670.

Growth Factors

To promote wound repair and/or tissue growth one or several substancescan be introduced into the present composite biocompatible materials orimpregnated into fabrics or prostheses made from the bifunctionalizedpolyester material. Exemplary substances include polypeptides such ashuman growth factors. The term “human growth factor” or “HGF” embracesthose materials, known in the literature, which are referred to as suchand includes their biologically-active, closely related derivatives. TheHGFs can be derived from naturally occurring sources and are preferablyproduced by recombinant DNA techniques. Specifically, any of the HGFswhich are mitogenically-active and as such effective in stimulating,accelerating, potentiating or otherwise enhancing the wound healingprocess are useful herein. Growth factors contemplated for use includeHEGF (urogastrone), TGF-beta, IGF, PDGF, and FGF. These growth factors,methods by which they can be obtained and methods and compositionsfeaturing their use to enhance wound healing are variously disclosed inU.S. Pat. Nos. 3,883,497; 3,917,824; 3,948,875; 4,338,397; 4,418,691;4,528,186, 4,621,052; 4,743,679 and 4,717,717; European PatentApplications 0 046 039; 0 128 733; 0 131 868; 0 136 490; 0 147 178; 0150 572; 0 177 915 and 0 267 015; PCT International Applications WO83/04030; WO 85/00369; WO 85/01284 and WO 86/02271, and UK PatentApplications GB 2 092 155 A; 2,162,851 A, and GB 2 172 890 A, all ofwhich are incorporated by reference herein.

The surface of the bifunctionalized polyester polymer of the inventioncan be modified with biologically-active proteins in order to emulatesome of the natural properties of native vessels, thereby improvinggraft patency and healing. For example, the enzymatic, chemotactic, andmitogenic properties of thrombin can be inhibited by surface bound rHir(recombinant hirudin). This inhibition can significantly reduce bloodproduct formation and maintain anastomotic smooth muscle cells in thequiescent state, thereby preventing the formation of anastomotic intimalhyperplasis. rHir has been shown to have potent anti-thrombin activitywhen covalently immobilized onto a DACRON® surface (see, e.g., PhaneufM. D., et al., Biomaterials 18(10):755 (1997) and Berceli S. A., et al.,J. Vasc. Surg. 27:1117 (1998)), or to another biomolecule (see, e.g.,Phaneuf M. D., et al., Thromb. Haemostas. 71(4):481 (1994)). Inaddition, covalent linkage of VEGF (vascular endothelial growth factor)may permit complete endothelialization of the graft surface by bothtrans-anastomotic and trans-membrane (through the remaining porosity)cellular migration. Techniques for binding growth promoting factors tobiocompatible materials are described in U.S. Ser. No. 09/139,507entitled “Growth-Promoting Biocompatible Substances and Methods of UseThereof,” and in Kubaska S. M. III, et al., Surgical Forum 49:322(1998), which are herein incorporated by reference.

Covalent linkage of protein to a biomaterial surface in order to createa “basecoat” layer has numerous beneficial advantages. Non-specific orcovalent attachment of a protein coating can “passivate” a surface thatis relatively thrombogenic, thereby decreasing adhesion of bloodproducts such as platelets, red blood cells, and fibrinogen (Rumisek J.,et al., Surgery 105:654 (1989)). Proteins incorporated as a basecoatlayer can be used as a “scaffolding” in order to promote a specificresponse such as linkage of RGD peptides to promote cell adhesion (LinH. B., et al. J. Biomed. Mater. Res. 345:170 (1994)). Additionally,increasing the angstrom distance between a biologically-active moleculeand the surface via polyethylene oxide groups can reduce sterichindrance on the target molecule, thereby maintaining activity (Park K.D., et al., J Biomed. Mater. Res. 22:977 (1988)).

Covalent linkage of a protein “basecoat” layer can serve as the spacerbetween rHir/VEGF and the biomaterial surface. Albumin can be used asthe basecoat moiety. Albumin, which is in natural abundance incirculating blood, has numerous beneficial results in vitro and in vivo(see, e.g., Kotteke-Marchant K., et al., Biomaterials 10:147 (1989) andPhaneuf M. D., et al., J Applied Biomater. 6:289 (1995)). Utilization ofa basecoat layer permits significant amplification of potential bindingsites for secondary protein attachment via heterobifunctionalcrosslinkers; thus creating a biomaterial surface with distinctproperties for a specific application. This technique has been used toprovide numerous binding sites for rHir, for example (see, e.g., PhaneufM. D., et al. ASAIO J. 44:M653 (1998) and Phaneuf M. D., et al.,Biomaterials 18(10):755 (1997)). Examples of other basecoat proteinsinclude, but are not limited to, collagen and fibronectin.Alternatively, the basecoat may be synthetic, such as, e.g., a Lys-Tyrmoiety or polyethylene oxide.

Additives/Modifiers

Additional modifiers that can be applied to the bifunctionalizedpolyester material include, but are not limited to, the following:thermally conductive agents (e.g., graphite, boron nitride),ultraviolet-absorbing agents (e.g., benzoxazole, titanium dioxide, zincoxide, benzophenone and its derivatives), water repellent agents (e.g.,alkylsilane, stearic acid salts), therapeutic agents (e.g., antibiotics,hormones, growth factors), stain resistant agents (e.g., mesitol,CB-130), rot resistant agents (e.g., zinc chloride), adhesive agents(e.g., epoxy-resin, neoprene), anti-static agents (e.g., amines, amides,quaternary ammonium salts), biocidal agents (e.g., halogens,antibiotics, phenyl mercuric acetate), blood repellents (e.g.,monoaldehyde urea resin), dye and pigments, electrically conductiveagents (e.g., metal particles, zinc oxide, stannic oxide, indium oxide,carbon black, silver, nickel), electromagnetic shielding agents (e.g.,hypophosphorous, carbon-phenol resin compounds), and flame retardantagents (e.g., aluminum hydroxide, borax, polyamide, magnesium hydroxide,polypropylene).

EXAMPLE 1

We tested whether we could generate amine functional groups on thesurface of polyester by treatment with ethylene diamine (EDA). Exposureof the polyester to EDA created both carboxylic and amine groups withinthe polymer structure as evidenced by uptake of both methylene blue(FIGS. 2A-2G) and acid red (FIGS. 32A-3G). Formation of these groupscould also be regulated by EDA concentration but was not significantlyaltered by the rinse time (see FIGS. 4A-4E for methylene bluedetermination of carboxylic acid groups and see FIGS. 5A-5E for acid reddetermination of amine groups). For the hydrolyzed material (HYD),carboxylic acid content decreased with increasing EDA concentrationwhereas amine content increased, suggesting amine groups were limited tothe outer periphery of the fiber. Amine content in the hydrolyzedsegments was not as elevated as the scoured segments (CNTRL). For theCNTRL and HYD polyesters, employing toluene as the solvent at lowerconcentrations increased carboxylic acid (see FIGS. 6A-6F for CNTRL andFIGS. 8A-8F for HYD) and amine (see FIGS. 7A-7F for CNTRL and FIGS.9A-9F for HYD) formation. In contrast to the water washing studies,exposing the segments to a prolonged toluene rinse increased formationof both carboxylic acid (FIGS. 10A-10G) and amine (FIGS. 11A-11G)functional groups.

Determination of Amine/Carboxylic Acid Content via Textile Dye Uptake

EDA exposure to scoured (CNTRL) and hydrolyzed (HYD) segments resultedin a yellowish-brown coloration as compared to unmodified CNTRL and HYDsurfaces, both of which remained white. Using acid red uptake, CNTRL-EDA(0.82±0.10 nmoles/mg) and HYD-EDA (0.32±0.02 nmoles/mg segments had 43-and 8-fold greater amine content as compared to their respectivecontrols. Amine formation was 2.6-fold greater using CNTRL as comparedto HYD material. Using methylene blue uptake, carboxylic acid content inthe CNTRL-EDA segments increased 18-fold whereas a 40-fold decrease incarboxylic acid content occurred for the HYD-EDA segments. Thiscarboxylic acid group loss in the HYD-EDA segments may be due to EDAreaction with the carboxylic acid groups created during the initialalkaline hydrolysis.

Determination of Primary Amine Functional Groups via Sulfo-SDTB

Sulfo-SDTB analysis of the control and EDA treated materials confirmedthe creation of amine groups into the polymer structure of the EDAtreated DACRON®. Additionally, amine content in the CNTRL-EDA (1.06±0.11nmoles/mg) and HYD-EDA (0.36±0.03 nmoles/mg) segments was comparable tothe results obtained in the dye uptake study.

Physical Characteristics of the EDA-Modified Polyester

Fiber weight loss from the CNTRL-EDA (2.3±0.55%) and HYD-EDA (1.3±0.25%)segments was 3.8- and 2.0-fold greater than their respective controls.The difference in fiber weight loss between HYD-EDA and CNTRL-EDAsegments (HYD-EDA segments lost 1.9-fold less fibers than the CNTRL-EDAsegments) could again be attributed due to EDA reaction with thecarboxylic acid groups previously created on the fiber surface viaalkaline hydrolysis thus restricting deep EDA penetration into thefiber.

Tensile strength of the CNTRL-EDA and HYD-EDA segments was decreased 1.7and 1.3 fold as compared to CNTRL and HYD segments, respectively.Ultimate elongation also followed a similar trend, with a 1.6 and 1.3fold loss in elongation in the CNTRL-EDA/HYD-EDA segments. Comparable tothe fiber weight loss study, HYD segments were less affected by exposureto EDA as compared to the CNTRL segments.

Accessible amine and carboxylic acid groups have been created within thepolymer backbone of both CNTRL and HYD polyester (e.g., DACRON®)materials as determined by dye uptake and sulfo-SDTB indicator.Additionally, the bulk physical characteristics of both materials stillremain.

Materials and Methods

Polyester Preparation: Segments (5 cm×5 cm) were cut from a large wovenfabric sample and washed in 500 ml scouring solution (10 g Na₂CO₃, 10 mlTween 20 in 1L double distilled water (ddH₂O)) for 30 minutes at 60° C.Samples were then rinsed in 500 ml ddH₂O for 30 minutes at 60° C.(CNTRL) and air-dried overnight. Some of these scoured segments werethen exposed to 500 ml of 0.5% NaOH at 100° C. for 30 minutes.Alternatively, other NaOH conditions ranging from 1-20% could also beemployed. These pieces were then rinsed with ddH₂O (room temperature)and air-dried overnight at room temperature.

Formation of Amine and Carboxylic Acid Groups: The primary procedureemployed for this study was to incubate a 5 cm×5 cm segment of eithercontrol or hydrolyzed polyester into 100% ethylene diamine (EDA, Sigma)for 80 minutes at room temperature. The segments were then removed andplaced into distilled water overnight (˜16 hours) at room temperature,followed by air-drying at 60° C. for 2 hours. Several other approacheswere performed. EDA concentration, rinse times and solvent type wereperformed for both control and hydrolyzed DACRON®.

Determination of Amine and Carboxylic Acid Content: Methylene blue, acationic dye, was employed to qualitatively determine carboxylic acidgroups within the EDA-exposed polyester segments. Briefly, a 500 mlstock solution (500 μg/ml) of methylene blue was prepared (80% Purity)in 0.1 M Tris-CL pH 8.0. A working solution of methylene blue wasprepared by aliquotting 1 ml of the stock solution and bringing to atotal volume of 100 ml with Tris buffer (5 μg/ml). Segments (1 cm²) werethen cut from scoured and hydrolyzed EDA segments. Working MB solution(4-10 ml) was added to each segment, and incubated for 1 hour on aninversion mixer. The segments were removed and placed into wash solutionconsisting of Tris buffer for one hour. Pre and post dye bath solutionswere read at 611 nm using Tris buffer as blank. Segments were thengrossly observed for color uptake and shade differences andphotographed. Carboxylic acid content (nmoles/segment) weight (mg) wascalculated using standard textile equations.

For amine content, acid red 1 (AR1), an anionic dye, was employed toquantitatively and qualitatively assess total (primary and secondary)amine content in the DACRON®-EDA segments. Briefly, a 500 ml stocksolution of AR1 (0.5 mg/ml, dye purity=60%) was prepared in 0.01 M MESpH 4.5 (MES). A working solution of AR1 was prepared by aliquotting 10ml of the stock solution and bringing to a total volume of 100 ml withMES buffer (50 mg/l). Segments (1.0 cm²) were cut from the respectivetreatments. Working AR1 solution (2-3 ml) was added to each segment andincubated for 1 hour on an inversion mixer. The segments were removedand placed into wash solution of MES buffer for one hour. Pre and postdye bath solutions were read at 530 nm using MES buffer as the blank.Segments were then grossly observed for color uptake and shadedifferences and photographed. Amine content (nmoles)/segment weight (mg)was calculated using standard textile equations.

Determination of Primary Amine Functional Groups via Sulfo-SDTB: A stockbuffer consisting of 50 mM sodium bicarbonate, pH 8.5 was prepared.CNTRL, HYD, CNTRL-EDA, and HYD-EDA segments (n=4/test condition; 1.0cm²) were cut and weighed. Sulfo-SDTB (3 mg) was weighed and dissolvedin 1 ml dimethyl formamide. After thorough mixing, the sulfo-SDTBsolution was brought up to a total volume of 50 ml with the stock sodiumbicarbonate buffer (working sulfo-SDTB solution). Stock buffer (1 ml)and 1 ml working sulfo-SDTB solution were added to each tube and reactedfor 40 minutes at room temperature on an orbital shaker. Segments werethen removed and washed for 10 minutes in 5 ml of distilled water on aninversion mixer. Immediately following the wash, 2 ml of a perchloricacid solution was added to each segment. Segments were reacted for 15minutes on the inversion mixer. The reaction solution (1 ml) was thenremoved and absorbance at 498 nm was measured. Using the extinctioncoefficient for sulfo-SDTB and the segment weight, amine content(nmoles/segment weight (mg) was determined.

Physical Characterization of EDA-Modified DACRON®: Fiber weight loss wasdetermined post-exposure to either distilled water (control) or EDA.CNTRL and HYD segments were prepared as previously described. Segments(4 cm²) were cut from each segment type (n=8 segments/treatment) andweighed. Half of the segments for each treatment were placed intodistilled water and the other half placed into 100% EDA for 80 minutes.All segments were then transferred to distilled water for 16 hours,followed by air-drying at 60° C. for 2 hours. Segments were thenreweighed, with the difference in segment weight determined.

Tensile strength and ultimate elongation were then determined. CNTRL,HYD, CNTRL-EDA, and HYD-EDA segments (width=1 inch, length=2 inches)were cut. A Q-test apparatus was calibrated at the time of use under acontrolled climate (room temperature—24.7° C., humidity—75%). A100-pound load cell was used and a pull rate of 12 inches/minute wasset. A gauge length of 0.75 inch was set into the apparatus, with atotal of 1.25 inches of each segment placed into the clamps. Stretchingwas then initiated and automatically stopped at the break of eachsegment. The peak load at break (1 lb) and the ultimate elongation foreach segment was determined.

EXAMPLE 2

We tested whether treatment of polyester fabric with amines other thanEDA would result in the generation of functional amine groups. Polyesterand hydrolyzed polyester were treated with four differentmultifunctional amines at a range of times and temperatures, and thendyed in diagnostic dyes. We specifically tested the uptake of CI AcidRed 1 or Methylene Blue dye by polyester fabric (hydrolyzed orunhydrolyzed) after treatment of the fabric at 85° C. with2-methylpentamethylene diamine (2MPD) for 10 minutes,tetraethylenepentamine (TEP) for 20 minutes, 1,2-diaminocyclohexane(12DC) for 2 hours, and 1,6-hexanediamine (16HD) for 24 hours. Theresults of these treatments are shown in FIG. 12. The loss in tensilestrength caused by these treatments is shown in Table 1. TABLE 1 Effectof diamines on fabric strength Amine/Treatment time @85 C. 2MPD TEP 12DC10 min 20 min 2 hr % strength loss Polyester 15 15 64 Hydrolyzed 0 0 20Polyester

While these amines differ in the ease of reaction with polyester(roughly similar effects occurring at times ranging from 10 minutes to24 hours), they are effective at providing amine groups at the fibersurface. It is notable that they also hydrolyze the surface and yieldcarboxylic acid groups (see below).

Treatment with EDA in Toluene

The effect of increasing EDA concentration when applied at constanttemperature and time (i.e., 20 hours at 50° C.) is shown in FIG. 13. Asexpected, increasing the applied concentration of EDA results in theformation of more amine groups on the fiber, as is determined by theuptake of C.I. Acid Red 1 dye, and the reaction seems thus to be readilycontrollable in the manner of dye application.

Treatment with EDA

For the sake of simplicity, the basic reaction of polyester (originaland hydrolyzed) in pure EDA at room temperature was used for themajority of the study to determine its effect on the fiber and itsproperties. The extent of treatment was controlled by the time ofimmersion in EDA. The gradual incorporation of amine groups at thepolyester surface was followed by dyeing with C.I. Acid Red 1. Thepresumed reaction scheme is shown in FIG. 1, reaction 3. FIG. 14 showsthe darkening of shade as treatment time increases. Noticeable is thatthe treatment on hydrolyzed material is less effective at generatingamine groups. Perhaps more surprising is the result of diagnostic dyeingin Methylene Blue. As expected, the hydrolyzed material has carboxylicacid groups present, but the treatment of unhydrolyzed material with EDAalso generates carboxylic acid groups. This is presumably due tohydrolysis involving the strongly basic diamine and any small amount ofwater present (FIG. 1, reaction 2). On treatment with EDA, the number ofcarboxylic acid groups in the hydrolyzed material initially decreases,and then increases again.

The quantification of these functional groups via dyeing with dyes ofknown purity produced the results shown in FIGS. 15A and 15B. Again, thegeneration of fewer amine groups and the initial loss of carboxylic acidgroups on the hydrolyzed material is shown.

An electron micrograph of untreated polyester and hydrolyzed polyester,and both after treatment with EDA is shown in FIGS. 16A-16D. It seemsapparent that aminolysis, as found in previous studies, is a morepenetrating treatment for polyester. Examination of these imagessuggests that the aminolysis, once started, proceeds more quickly in theinitial areas of attack: areas of cracking are isolated among apparentlyundamaged material. Avny and Rebenfeld postulated an “induction” periodand a subsequent “autoaccelerated” reaction (J. Applied Polymer Science32:4009, 1986). Cracking is also visible on extensively treated fibersusing visible microscopy, and cross-sections of the diagnostically dyedfibers show the treatment to be confined to the fiber surface. Theslowing of aminolysis by a previous hydrolysis is also apparent when theeffect of this treatment on the tensile strength of the material isconsidered. FIG. 17 shows the change in tensile strength with time ofEDA treatment. After an initial drop (comparable with the loss ofstrength on hydrolysis alone) the tensile strength falls more slowly asthe treatment continues. This again seems to suggest that the effect ishappening more quickly on a few areas, rather than very generally.Weight loss data supported this contention (Table 3): only aftercomparatively long treatment times is a significant weight loss noted.TABLE 3 Weight Loss on EDA Treatment EDA Treatment Weight loss Time(min) (%) Polyester 80 0.85 120 3.17 Hydrolyzed 80 0.15 Polyester 1200.33

Efforts to determine the absorbency (wetting time) and static propertiesof these EDA-treated material were unsuccessful: all materials gaveresults that varied widely. The Soil Release ratings did, however,indicate that the EDA treatment has an effect (Table 4). TABLE 4 SoilRelease Ratings of Treated Polyester SR Rating Untreated 3.63 Hydrolyzed4.37 EDA-treated 4.13 Hydrolyzed/EDA treated 4.94

All treatments produced an improvement over untreated polyester.Alkaline hydrolysis, as previously established, gives an improvement insoil release. EDA treatment alone was less beneficial. A combination ofhydrolysis and EDA treatment, however, gave excellent soil releaseproperties.

The greater hydrophilicity of surface that the increased soil releaseproperties imply is also represented in the wicking data (FIG. 18). Thehydrolyzed surface wicks water at a greater rate when distance ismeasured. However, when the weight of water is considered, at longertreatment times the EDA treated material takes up a slightly greaterweight of water; this is possibly due to the greater access to the fiberinterior allowed by the surface cracking. In both cases there is asuggestion that at shorter times of EDA treatment, the wickingperformance is reduced.

The treatment of polyester with bifunctional aliphatic amines,especially ethylene diamine, generates amine groups on the fibersurface, as expected. Somewhat surprisingly, the reaction results in thesimultaneous formation of carboxylic acid groups in a manner akin to thefamiliar alkaline hydrolysis. The reaction is slower when applied topolyester that has previously been subjected to alklaline hydrolysis.The reaction is readily controllable, and when applied to untreated orhydrolysed polyester has the potential to provide polyester surfaceswith varying levels of amine and carboxylic acid functionality. Thetreatment is of great potential use in modifying the biomedicalproperties of polyester, and will allow for the binding of differentbiologically-active agents (e.g. anti-thromboytic agents (e.g.,anti-thrombin), thrombolytic agents, growth-promoting/inhibiting agents,antimicrobial or antifungal agents) to give multifunctional materials.

As would be expected from a more hydrophilic surface, wicking and soilrelease are improved by the treatment.

Materials and Methods

Polyester Material: A plain weave 100% polyester fabric was used in allexperiments (Style 755, Testfabrics, Inc., West Pittston Pa.)

Chemicals: Chemicals used were laboratory grade, including ethylenediamine, (“EDA”,99%) and sodium hydroxide. All water used in theexperiments was de-ionized. Methylene Blue was obtained in 99% purity.CI Acid Yellow 4, previously synthesized in our laboratory, was purifiedfrom salt impurities by extraction in N,N-dimethylformamide.

Apparatus: An Ahiba Polymat (Datacolor International) dyeing machine wasused in the hydrolysis treatment: other reactions were carried out insimple glassware. A Cary 50 UV-Visible Spectrophotometer, Varian PtyLtd, was used in measuring dye uptake. A Qtest CRE (Constant Rate ofExtension) tester, MTS Systems Corporation was applied to determine thetensile property. A Joel 5900 Scanning Electronic Microscope was used toexamine the morphological modification on the fiber surface.

Fabric Treatment: Polyester fabric was subjected to alkaline hydrolysisby treatment in 1.0% w/v sodium hydroxide for 1 hr at 98° C., LR 40: 1,followed by rinsing in water and air-drying. These conditions wereearlier found to provide surface carboxylic acid functionality withminimal strength and weight loss. Treatments in bifunctional amines werecarried out under a range of conditions.

Multifunctional amines 1,6-hexanediamine (16HD), 2-methylpentamethylenediamine (2MPD), 1,2-diaminocyclohexane (12DC) and tetraethylenepentamine(TEP) were applied to polyester at 100% concentration (at 10:1 liquorratio) in glass vials at a range of temperatures and times in alaboratory oven. Ethylene diamine was applied to untreated polyesterfrom a range of solution concentrations in toluene. Specimens wererinsed in acetone and then water, and dried.

Both untreated and hydrolyzed polyester fabric specimens were treated byimmersion flat in ethylene diamine (10:1 LR) at room temperature (25°C.) for a range of times. After each treatment, specimens were removedand rinsed in de-ionized water until the water reached neutral, and airdried. Fabrics were conditioned under standard conditions for 24 hoursbefore physical tests.

Tests: The following tests were carried out on treated and untreatedfabrics:

1. Surface functional groups were determined by dye uptake experiments.For simple visual analysis, carboxylic acid groups were visualized bythe uptake of Methylene Blue from a 50:1LR bath of 0.1 g/l solution in 1g/l ammonia, with a treatment at 60° C. for 20 minutes. Amine groupswere visualized by dyeing in C.I. Acid Red 1 (0.1 g/l in 1 g/l aceticacid, 50:1 LR, 60° C., 20 min).

For more accurate quantification, dyeing with Methylene Blue was carriedout with 0.25% owf of dye, 50:1 liquor ratio, with temperature set to40-50° C. for 50 minutes. Ammonium hydroxide was used to adjust the dyebath to pH 9.5. Amine groups were quantified by the uptake of purifiedCI Acid Yellow 4 under the same conditions, except that the pH wasadjusted to 4.0 with acetic acid. This dye was chosen since it is ofknown formula and is monosulfonated (FIG. 11).

After dyeing, solutions were diluted to a convenient concentration andtheir absorbance measured. Dye uptake was calculated by reference topre-established absorbance/concentration relationships.

The amino or carboxylic acid groups were quantified using the followingequation:Functional Group Density=(Q/M)/W

Where Q represents the amount of dye taken up, M is the molecular weightof the Dye, and W is the weight of the fabric.

2. Weight Change was determined by measuring the constant oven-driedweight of fabrics before and after aminolysis and is expressed inpercentage. Specimens in this test were pre-raveled 1 cm from the edgesto avoid weight loss caused by raveling of yarns from fabric duringtreatment and rinsing:

3. Tensile Loss was measured on 25×150 mm raveled fabric stripesobtained from the warp direction of conditioned fabric. Using acrosshead speed of 200 mm/min and gage of 75 mm, breaking load andelongation of specimens was determined. % Loss of tensile strength wascalculated.

4. Wicking Properties were assessed in terms of both the rate and amountof water wicked. Specimens of25×150 mm were weighted at one end andimmersed to a depth of 10 mm in a beaker of water on an analyticalbalance (0.1 mg; FIG. 12). The weight of the beaker was recorded everyminute over a ten-minute period, and the average rate of wicking(mg/min) calculated. In addition, the height to which the wicked waterreached was measured, and the average height per five minutescalculated.

5. Wetting Time was measured using AATCC TM 79-2000 (Absorbency ofbleached textiles).

6. Electrostatic Cling was measured using AATCC Method 115-2000.

7. Soil Release Properties were assessed using AATCC Test Method130-2000.

EXAMPLE 3

We next sought to determine whether the generation of carboxylic acid oramine functional groups on polyester could be used to provide potentialindividual “anchor” sites for covalent attachment of biologically-activeproteins. To address this issue, we modified polyester (DACRON®) as isdescribed herein and quantified the protein binding to the carboxylicacid and amine groups on the surface.

Woven DACRON® patches (1 cm²) were treated with EDA for 80 minutes at25° C. Patches were divided into three groups: untreated DACRON® (CTRL),control-EDA (C-EDA) DACRON®, and Tr-EDA DACRON® (EDA-treated DACRON®reacted with Traut's Reagent, a heterobifunctional crosslinker thatreacts with primary amine groups on the surface). Bovine serum albumin(BSA, 1 mg) was radiolabeled with ¹²⁵I. BSA was then reacted with theheterobifunctional crosslinker Sulfo-SMCC for 20 minutes at 37° C. Eachgroup of patches was then incubated on an orbital shaker for 3 hours at25° C. with ¹²⁵I-BSA-Sulfo-SMCC.

A second study involved CTRL and C-EDA segments as well as EDC-EDAsegments (EDA-treated patches reacted with EDC, a carbodiimidecrosslinker that reacts with carboxylic acid groups on the surface). BSA(1 mg) was radiolabeled with ¹²⁵I-BSA. Each group of patches was thenincubated with ¹²⁵I on an orbital shaker for 3 hours at 25° C.

A third study, which again involved CTRL and C-EDA patches, alsoassessed Tr-EDC-EDA patches (EDA-treated DACRON® patches reactedsimultaneously with the Traut's Reagent and EDC). BSA (1 mg) wasradiolabeled with either ¹²⁵I or ¹³¹I. The ¹²⁵I-BSA was again reactedwith Sulfo-SMCC. Each group of patches was then simultaneously incubatedwith ¹²⁵1-BSA-Sulfo-SMCC and ¹³¹1-BSA on an orbital shaker for 3 hoursat 25° C. For each study, patches were washed in detergent andsonicated, followed by gamma counting. Lowry and TCA assays wereperformed to assess BSA concentration and radiolabeling efficiency,respectively. Using this data, specific activity for the BSA samples wascalculated. Protein binding was then determined as amount of protein(ng) per DACRON® segment weight (mg).

Albumin binding, which was either non-specific or covalent, occurred onall of our surfaces. For the first single binding study, ¹²⁵I-BSAbinding to the Tr-EDA group (360±10 ng/mg) was 20.4 fold and 2.3 foldgreater than CTRL (1.8±0.3 ng/mg, p=2.5×10⁻⁸) and C-EDA (155±3 ng/mg,p=8.8×10⁻⁷) segments, respectively (FIG. 20).

For the second single protein binding study, ¹²⁵I-BSA binding to the.EDC-EDA segments (184±6 ng/mg) was 2.9 fold and 1.5 fold greater thanCTRL (64±3 ng/mg, p=1.15×10⁻⁶) and C-EDA (123±2 ng/mg, p=4.79×10⁻⁵)segments, respectively (FIG. 21).

For the double protein binding study, ¹²⁵I-BSA and ¹³¹I-BSA binding tothe Tr-EDC-EDA segments (367±6 ng/mg and 286±8 ng/mg, respectively) was26.5 fold and 11.5 fold greater than CTRL segments (14±1 ng/mg,p=1.5×10⁻⁹; 25±5 ng/mg, p=1.37×10−4, respectively), and 2.9 fold and 3.1fold greater than the C-EDA segments (127±6 ng/mg, p=1.21×10⁻⁷; 94±5ng/mg, p=4.5×10⁻⁷, respectively; FIG. 22).

Reaction of EDA with DACRON& provides functional groups within thepolymer backbone. These functional groups are accessible for eitherindividual or simultaneous protein binding and can be used for covalentattachment of biologically-active proteins to the DACRON® surface.

EXAMPLE 4

In Vitro and In Vivo Assessment of Novel Bifunctionalized DACRON®Surfaces

We next sought to evaluate endothelial cell proliferation on DACRON®surfaces modified with the bifunctional amine ethylene diamine (EDA) invitro and to assess the wound healing response to the modifications invivo.

In Vitro

MYLAR®, a flattened form of DACRON®, was used for in vitro experiments.Discs (1.5 cm diameter) were treated with either 15% NaOH for 30 minutesat 100° C. (HYDRO), EDA for 80 minutes at 25° C. (C-EDA), or acombination of NaOH and EDA (H-EDA) to create functional groups. Humanumbilical vein endothelial cells (HUVEC) were then cultured with eithercomplete media or serum-starved media on our modified MYLAR® surfaces.Untreated MYLAR® served as the control (CTRL). HUVECs added to tissueculture wells without MYLAR® were also monitored for cell viability.HUVEC growth was monitored at 1, 2, 3, and 4 days using an Alamar blueassay. Alamar blue interacts with the cell wall of live cells and can bedetected at a fluorescence emission spectra of 590 nm.

HUVEC proliferation occurred on all of our modified surfaces throughoutthe four-day time interval. H-EDA surfaces demonstrated significantlygreater HUVEC growth with complete media as compared to the CTRLsurfaces (64.05±4.38 vs. 38.25±10.68, p=0.03). C-EDA surfacesdemonstrated significantly prolonged HUVEC life with serum-starved mediaas compared to the CTRL surfaces (3.30±0.51 vs. 1.57±0.28, p=0.005).

In Vivo

For the in vivo study, woven DACRON® patches (1 cm²) were treated usingthe same methods as employed for the MYLAR® discs. The patches were thenimplanted into a subcutaneous rat model for 14 days, explanted, andwound healing assessed using histological techniques.

Histological evaluation of our explanted patches revealed no impairmentor overall difference in wound healing between the modified DACRON®patches and CTRL.

This study demonstrates that covalent attachment of biologically-activeagents, such as growth factors, to the accessible functional groups onthis DACRON® surface can be used to promote endothelial cell recruitmentfollowing, e.g., prosthetic arterial grafting.

Other Embodiments

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

Other embodiments are within the claims.

1. A composition comprising a polyester polymer functionalized tocomprise a chemical group capable of forming a covalent bond with afirst compound.
 2. The composition of claim 1, wherein said compositionis bifunctionalized to comprise a first chemical group and a secondchemical group.
 3. The composition of claim 2, wherein said firstchemical group is a carboxylic acid group and said second chemical groupis an amine group.
 4. The composition of claim 1, wherein said chemicalgroup comprises a carboxylic acid group.
 5. The composition of claim 1,wherein said chemical group comprises an amine group.
 6. The compositionof claim 1, wherein said polyester material is functionalized bytreatment with ethylene diamine (EDA).
 7. The composition of claim 6,wherein said polyester material is treated by alkaline hydrolysis priorto treatment with EDA.
 8. The composition of claim 1, wherein said firstcompound comprises a chemical compound or a biologically-active agent.9. The composition of claim 8, wherein said chemical compound comprisesa commercial finish selected from the group consisting of flameretardants, repellents, antistatic agents, and dyes.
 10. The compositionof claim 8, wherein said biologically-active agent comprises anantimicrobial agent, an antifungal agent, an anti-thrombolytic agent, athrombolytic agent, an antiviral agent, an antiseptic agent, agrowth-promoting agent, or a growth-inhibiting agent.
 11. Thecomposition of claim 8, wherein said biologically-active agent comprisesa peptide, a polypeptide, a nucleic acid molecule, an antibody, or asmall molecule.
 12. The composition of claim 11, wherein saidpolypeptide comprises anti-thrombin, fibronectin, fibrinogen,vitronectin, collagen, streptokinase, urokinase, tissue plasminogenactivator (tPA), vascular endothelial growth factor (VEGF), orgamma-interferon.
 13. The composition of claim 12, wherein saidanti-thrombin is hirudin.
 14. A biocompatible device comprising abifunctionalized polyester polymer, wherein said bifunctionalizedpolyester polymer comprises a biologically-active agent that iscovalently bonded to said polymer via a carboxylic acid group or anamine group.
 15. The device of claim 14, wherein said device is selectedfrom the group consisting of a catheter, a vascular graft, an artificialheart, a blood filter, a pacemaker lead, a heart valve, and a prostheticgraft.
 16. A wound dressing comprising a bifunctionalized polyesterpolymer covalently bonded to a biologically-active agent via acarboxylic acid group or an amine group.